USP <85> Bacterial Endotoxin Validation and Its Importance to the Test Method

Evelyn Orona, ARL Bio Pharma Associate Microbiology Laboratory Supervisor

Andrew Taylor, ARL Bio Pharma Microbiology Laboratory Supervisor

ARL Bio Pharma utilizes the kinetic turbidimetric method described in USP <85> Bacterial Endotoxins Test to provide results. An endotoxin test detects toxins that are released from the cell wall of disrupted gram-negative bacteria. This is a reaction over time test that quantitates the amount of endotoxin in a sample compared to a standard curve. This article discusses USP <85> challenges and why method validation is important to provide consistent results.

The most common challenge encountered while performing endotoxin testing is that many drug products cause the assay reaction to slow down (inhibition) or speed up (enhancement) during the testing process, which does not allow for an accurate result to be obtained. Those properties which cause the inhibition or enhancement must be overcome to obtain a reliable and repeatable test result. Microbiologists performing testing must determine the degree in which inhibition or enhancement is occurring. This is done by adding known amounts of endotoxin to test samples and comparing the results to those known values. If there is no inhibition or enhancement, results from the test samples spiked with a known amount of endotoxin will be the same as the expected results. Inhibition would artificially lower results for the test sample, and enhancement would increase results for the test sample, relative to the expected spike concentration. 

Once inhibition or enhancement is understood, microbiologists can develop a plan of action to overcome those sample properties.

For sample testing, overcoming inhibition or enhancement typically begins with dilution of the sample in endotoxin-free water. A test sample cannot be diluted so much that the test results obtained become an inaccurate representation of any endotoxin that is present. A sample's Maximum Valid Dilution calculated from the sample's endotoxin limit (provided by the client or calculated using the route of administration, the maximum dose per hour, and the average patient weight) allows microbiologists to design endotoxin testing using the appropriate sample dilution. Additional reagents may also be added to overcome inhibition or enhancement if an appropriate method cannot be obtained through dilution alone.

Endotoxin validation testing provides a more robust assessment of the test method. Briefly, inhibition and enhancement testing are performed using multiple dilutions/diluents simultaneously. The results are then assessed to see which method generates the results closest to the ideal spike recovery percentage and sample pH. Once the appropriate sample preparation steps are determined, the sample is tested in triplicate using the chosen method. Should this method provide accurate and precise test results, it is considered validated and retained in ARL's method suitability database for future submissions of the product. This ensures this method will be used for every submission of a particular formulation.

Once an appropriate method is determined, the lowest value on the standard curve is used to calculate the concentration of endotoxin in a sample, should any be present. For samples showing the presence of endotoxin, the software used by ARL (EndoScan-V by Charles River) will calculate a numerical value that will be reported. For samples showing no presence of endotoxin, the software generates a "less than" numerical value. This "less than" numerical value is calculated by multiplying the dilution factor used by the lowest point on the standard curve (for samples using mL units) or by dividing the lowest point on the standard curve by the test concentration (for samples in all other units). Where no presence of endotoxin is detected, the software cannot calculate an exact numerical value, thus a "less than" value is reported.

When no validated method is on file, microbiologists have the discretion to choose a sample dilution to use for each sample test. This creates the potential for different dilutions to be used between sample submissions, which will lead to different results being reported. While this complies with USP <85> guidelines, many clients choose to perform Endotoxin Validation on formulations submitted for endotoxin testing to have consistent testing methods and results.

For more information on USP <85> Endotoxin Testing and Method Validation, contact 800-393-1595 or


  • USP <85> Bacterial Endotoxins Test